Mutation of R2 does not interfere with virion entry or disassembly in axons.

(A) DRG sensory neuron explants were loaded with the CCF2 beta-lactamase (β-Lac) substrate and infected with either wild-type (WT) or R2-mutant (R2) PRV encoding β-Lac fused to the pUL35 capsid protein, or wild-type PRV not encoding β-Lac. As an added baseline control, the wild-type (β-Lac) virus was incubated with Accutase for 2 hours to prevent infection by removing the viral glycoproteins. Ratiometric image pairs were captured at 460 nm and 530 nm using 405 nm for excitation. β-Lac cleavage of CCF2, resulting from virion entry into the cells, was monitored as an increase in the 460:530 nm ratio. Data points represent the average fluorescence intensity ratio from > 60 axons (3 independent experiments each consisting of measurements from 20–30 axons per time point). Values are mean ± s.d. (B) Fusion-based entry was indirectly monitored by the disassociation of envelope and outer tegument proteins from capsids using dual-fluorescent tagged recombinants of PRV and HSV-1. Capsids were scored for coincident red/green emissions from extracellular virions (open bars) or from particles moving in axons at 3.5 hpi (solid bars). For the latter, only capsids moving > 2.5 μm were scored, and the percentage of particles that colocalized with eGFP was tallied across three independent experiments with > 3 fields imaged per experiment. Values are mean ± s.d.